MCB 120 Exam 1A
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MICROBIOLOGY 120. Microbial Physiology
Course description: Physiological processes in microorganisms including a study of structure, energy production, macromolecular biosynthesis, nutrition and growth
Credits: 3 units (3 hrs of lecture per week)
Prerequisite: MCB 1 and CHEM 160
Course Objectives:1) To identify the structure, chemistry and functions of major structures in a bacterial cell;2) To elucidate the requirements of microbial growth;3) To identify the various metabolic pathways present in different
microorganisms;4) To understand enzyme regulation present in microbial cells;5) To relate the principles of microbial physiology to the industrial
applications of microorganisms.
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OUTLINE
I. Introduction
II. The bacterial cell: structures and functions
1. Major cellular structures
2. Chemistry and synthesis of cellular structures
First long exam
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III. Microbial Growth
1. Definition of growth2. Measurements of growth3. Growth Physiology4. Steady-state Growth and Continuous5. Factors affecting growth
IV. Microbial Nutrition and Solute Transport
V. Bioenergetics in the Cytosol
Second long exam
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VI. Bacterial Metabolism
1) Central metabolic pathways
a) Glycolysisb) Pentose Phosphate Pathwayc) Entner-Doudoroff pathwayd) Tricarboxylic Acid Cyclee) Glyoxylate Cycle
Third long exam
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2. Fermentation3. Photosynthesis4. Metabolism of lipids, nucleotides, amino
acids and hydrocarbon5. Inorganic metabolism6. C1 metabolism
Fourth long exam
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ATLAS, R.M. 1995. Principles of Microbiology. Mosby-Year Book Inc., Missouri.
MADIGAN, M., MARTINKO, J. and PARKER, J. 2012. Brock Biology of Microorganisms.13th edition. Prentice Hall, New Jersey.
NEIDHART, F.C., INGRAHAM, J.L. and SCHAECHTER, M. 1990. Physiology of theBacterial Cell. A Molecular Approach. Sinauer Associate, Inc., Massachusetts.
STRYER, L. 1995. Biochemistry. W.H. Ferman and Co., New York.
WHITE, D. 2007. The Physiology and Biochemistry of Procaryotes. Oxford UniversityPress, New York
Specific journal articles as cited
On-line materials as cited
REFERENCES
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1.0 95.6 - 1001.25 91.1 - 95.51.5 86.7 - 91.01.75 82.2 - 86.62.0 77.8 - 82.12.25 73.3 - 77.72.5 68.9 - 73.22.75 64.4 - 68.83.0 60.0 - 64.34.0 55.0 - 59.95.0 <55.0
GRADING SCALE
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ATTENDANCE AND MAKE-UP EXAM
1. A general make-up exam which is comprehensive in scope will be given towards theend of the semester to students who missed any of the 4 long exams due to a validreason (an official excuse slip must be presented).
2. A student who incurs at least 10 absences in the lecture will be automatically droppedfrom the course. Attendance sheets will be passed around for monitoring of thestudent's attendance in class.
If majority of the absences are excused, the student shall be given a grade of DRP.
If majority of the absences are unexcused, the student shall be given a grade of 5.0.
3. Excuse slips must be presented not later than the second class session following thestudent's return.
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LECTURER’S INFORMATION
Name: DR. RINA B. OPULENCIA
MS Microbiology: University of Queensland, Australia
PhD Microbiology: University of Illinois, Urbana-Champaign, USA
Office: B-302 or D-333
Consultation hours:WED and FRI: 9:00 AM-11:00 AM; 2:00 PM - 4:00 PM
TUES: 2:00 PM - 4:00 PM
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Physiology- study of the functions of living organisms and their physicochemical parts and metabolic reactions
cytology (physical and chemical structures) biochemistry (enzymes and chemical reactions) nutrition genetics
Microbial Physiology- study of life processes of microorganisms
INTRODUCTION
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1. Knowledge of microbial physiology can be applied toother fields.
2. Microorganisms can serve as models to understand lifeprocesses.
Importance of Studying Microbial Physiology
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Importance of Prokaryotes
ubiquitous
exhibit great metabolic and genetic diversity
comprise the majority of organic matter
major environmental determinants on earth
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Whitman, W. B. 1998. PNAS USA. 95: 6579
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Whitman, W. B. 1998. PNAS USA. 95: 6579
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White, D. 2006Whitman, W.B. 2009. J. Bacteriol. 491:2000-2005
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Lipids: alcohols ether-linked toglycerol Cell Wall: variable, some havepseudo-PG Genome: eukaryotic-typehistones; DNA organized intonucleosome-like structures Transcription machinery: RNAPhas 8-10 subunits, like eukaryotes;RifR
Lipids: fatty acids ester-linkedto glycerol Cell Wall: Peptidoglycan (PG) auniversal component Genome: histone-like proteins,but not organized like nucleosomes
Transcription machinery: RNAPhas 4 subunits; is RifS
ARCHAEA vs BACTERIA
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Translation machinery: Use Met as initiator amino acid;insensitive to translational inhibitorsthat affect bacteria; require EF-2like eukaryotic ribosomes
UNIQUE: Light-driven ion pumps (halophiles) Unique coenzymes (methanogens)
Translation machinery:Use f-Met as initiatoramino acid; sensitive totranslational inhibitors,e.g., Tet, Cm
ARCHAEA vs BACTERIA
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MICROBIOLOGY 120. MICROBIAL PHYSIOLOGY
majority of the topics is “bacterial” physiology
studies done on Escherichia coli or Bacillus subtilis
Model organisms provide basis for understandingimportant biological principles but not all bacteria are the same.
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The Prokaryotic Cell
Brock Biology of Microorganisms 10/eMadigan/Martinko/Parker
2003 Benjamin Cummings
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CELL WALL
fairly rigid layer that lies outside the plasma membrane
Importance:- confers shape- protects the cell from osmotic lysis- anchors the flagellum- adds to pathogenicity of the cell- protects the cell from toxic substances and pathogens
Bacteria can be divided into two big groups based on cell wall structure.
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BACTERIAL CELL WALL
Brock Biology of Microorganisms 10/eMadigan/Martinko/Parker
2003 Benjamin Cummings
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GRAM POSITIVE CELL WALL
Characteristics
A. Thick layer of peptidoglycan (murein; mucopeptide)
•• a polymer of disaccharide linked by polypeptidea polymer of disaccharide linked by polypeptide
•• insoluble, porous, big polymerinsoluble, porous, big polymer
•• > 50% of the cell wall> 50% of the cell wall’’s dry weight; 15 - 40 nm thicks dry weight; 15 - 40 nm thick
•• isolatable as murein sacculusisolatable as murein sacculus
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Peptidoglycan Subunit
Brock Biology of Microorganisms 10/eMadigan/Martinko/Parker
2003 Benjamin Cummings
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Peptidoglycan Interbridge
Type I. Direct D-alanyl-R3 peptide bond - found in E. coli and other gram negative bacteria - also found in many bacilli
Type II. Pentaglycine or Other L- or D- amino acid sequences - varies from organism to organism
Type III. A bridge composed of one to several peptides, each having the same amino acid sequence as the peptide unit attached to muramic acid
Type IV. A bridge extending between carboxyl groups belonging to either D-alanine or D-glutamic acid and a diamino acid residue or a diamino acid containing short peptide
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Peptidoglycan Interbridge
Brock Biology of Microorganisms 10/e
Madigan/Martinko/Parker
2003 Benjamin Cummings
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Peptidoglycan Interbridge
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Peptidoglycan Polymer
Brock Biology of Microorganisms 10/e by Madigan/Martinko/Parker
2003 Benjamin Cummings
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Enzymes
1 - glutamine:fructose-6-P aminotransferase 2 - glucosamine-P transacetylase 3 - N-acetylglucosamine phosphomutase 4 - UDP-N-acetylglucosamine pyrophosphoryalse
Peptidoglycan Synthesis: Synthesis of UDP-derivatives
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Peptidoglycan SynthesisEnzymes
1 – enoylpyruvate transferase2 – UDP- N-acetylpyruvoylglucos- amine reductase3 – each amino acid is added by
separate enzyme
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http://micro.digitalproteus.com/pics/peptidoglycansynthesis.jpg
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Synthesis of peptidoglycanoccurs in three phases:assembly of precursor in thecytoplasm, transport acrossthe inner membrane, andpolymerization. The lipid-linked muropeptide (lipid I)is generated in thecytoplasm from amino acidsand UDP-MurNAc (MurNAcis depicted by orangesquares). Transfer of N-acetylglucosamine (bluesquares) from UDP-GlcNAccompletes formation of theprecursor lipid II.Translocation across theinner membrane occurs,and subsequently, the chainpolymerizes while attachedto the lipid carrier. The unitis then transferred toexisting peptidoglycan.(PEP)Phosphoenolpyruvate; (m-DAP) meso-diaminopimelicacid.
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=glyco2&part=ch20
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B. Presence of Teichoic acids
- polymers of repeating units of glycerol or ribitol joined by phosphates
- amino acids (D-ala) or sugars (glu) are attached to glycerol/ribitol
- covalently linked to murein through muramic acid
- connected/embedded in PG layer or to membrane lipids
GRAM POSITIVE CELL WALL
lipoteichoic acid
- linear polymers of 16-40 phosphodiester-linked glycerophosphateresidues covalently linked to the cell membrane
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GRAM POSITIVE CELL WALL: TEICHOIC ACID
Brock Biology of Microorganisms 10/e by Madigan/Martinko/Parker
2003 Benjamin Cummings
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1. highly antigenic
2. anchors the wall to the cell membrane
3. provides high density of regularly oriented charges
4. storage of phosphorus
5. facilitates attachment of bacteriophage
6. inhibits activity of autolytic enzymes which hydrolyze the murein
TEICHOIC ACID: Properties and Importance
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TEICHOIC ACID: Variations
A. Glycerol Teichoic Acids
1. –Glycerol- P
2. –Glycerol- Palanyl
3. –Glycerol- Pglucosaminyl
alanyl
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TEICHOIC ACID: Variations
B. Ribitol Teichoic Acids
P7. –ribitol- P 8. –ribitol-
ala glucosyl
P 9. –ribitol-
ala NAG
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Teichuronic acids – acidic polysaccharides containing uronic acids
Neutral polysaccharides – important in classification of some Gram (+)
Other glycolipids – may substitute for whatever function of the LTA
Mycolic acids – waxy lipids found in Mycobacterium
Other substances which may be found on the cell wall:
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Substances active against peptidoglycan synthesis
1. Phosphonomycin (Fosfonomycin)- prevents synthesis of UDP-NAM from UDP-NAG
2. Cycloserine- inhibits the formation of the pentapeptide
3. Bacitracin - inhibits incorporation of lysine into the peptidoglycan- prevents dephosphorylation of the carrier lipid
4. Vancomycin, Tunicamycin, Ristocetin- inhibits translocation step of peptidoglycan
5. Penicillin - prevents cross-linking
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Beta-lactam antibiotics
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Beta-lactam antibiotics
Brock Biology of Microorganisms
10/e Madigan/Martinko/Parker
2003 Benjamin Cummings
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Beta-lactam antibiotics
Mode of action:
inhibition of final stages of assembly of the peptidoglycan
• must penetrate the cell wall and operate at theperiplasmic space
bind to “penicillin-binding” proteins in the outer leaf of the cell membrane
• enzymes which are responsible for the final stages of assembly of the peptidoglycan molecule
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not knownnot knownnot known1B1C, 7, 8
Destruction ofunutilized pentapeptide
DD-carboxypeptidase6006
Destruction ofunutilized pentapeptide
DD-carboxypeptidase1,8005
Cross-link hydrolysisduring cell elongation
DD-endopeptidase,DD-carboxypeptidase
1104
murein synthesisduring septation
transglycosylase-transpeptidase
503
growth in rod shape;cell elongation
transpeptidase202
murein synthesisduring cell elongation
transglycosylase-transpeptidase
100 (each)1A or 1B
Possible FunctionKnown EnzymeActivities
Moleculesper cell
PBP
Properties of penicillin-binding proteins
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GRAM NEGATIVE CELL WALL
1. Peptidoglycan
- thin: 1-2 layers in E. coli ( 2 - 6 nm thick)- constitute not more than 5 – 10% of wall’s dry weight- may be more of a gel than a compact layer
2. Outer membrane
- located above/external to peptidoglycan layer- like the cytoplasmic membrane- other main components
a. lipopolysaccharides (LPS)b. lipoproteinsc. outer membrane proteins (porins)
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GRAM NEGATIVE CELL WALL
Brock Biology of Microorganisms 10/e by Madigan/Martinko/Parker
2003 Benjamin Cummings
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GRAM NEGATIVE CELL WALL:
LIPOPOLYSACCHARIDES
- lipids and carbohydrates
- outer layer of the outer membrane
- endotoxin
- consists of three parts:
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GRAM NEGATIVE CELL WALL:
LIPOPOLYSACCHARIDE LIPID A
- embedded in the membrane as part of the lipid bilayer- hydrophobic- composed of 2 glucosamine residues linked β-1,6
(backbone) with four identical fatty acids
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GRAM NEGATIVE CELL WALL:
LIPOPOLYSACCHARIDE CORE
Outer core- shows high to moderate variability- consists of hexoses
Inner core- shows low structural variability- consists of 2-keto-3-deoxyoctonate (KDO),
heptose, ethanolamine and phosphate
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GRAM NEGATIVE CELL WALL:
LIPOPOLYSACCHARIDE O-ANTIGEN
- short polysaccharide extending outward from the core- consists of peculiar sugars which varies between bacterial
strains- not essential for viability
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GRAM NEGATIVE CELL WALL:
LIPOPOLYSACCHARIDE
Brock Biology of Microorganisms 10/e
Madigan/Martinko/Parker
2003 Benjamin Cummings
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GRAM NEGATIVE CELL WALL:
LIPOPOLYSACCHARIDE
Importance:
avoidance of host defenses (O-antigen)
contributes to the negative charge on the cell’s surface
stabilizes membrane structure
acts as endotoxin
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GRAM NEGATIVE CELL WALL:
LIPOPROTEIN
Brock Biology of Microorganisms 10/e Madigan/Martinko/Parker
2003 Benjamin Cummings
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GRAM NEGATIVE CELL WALL:
LIPOPROTEIN
- mediate interconnection between the OM and murein
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OUTER ENVELOPE: PORINS
form small hydrophilic channels through the outer envelopeallowing the diffusion of neutral and charged solutes ofMW <600 daltons
three identical units
associate to form membrane holes
transmembrane
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Anion-selective diffusion channels in Pseudomonas;induced under phosphate limitation
Protein P
Specific porin for maltose, maltodextrinReceptor for bacteriophage λ
LamB
Anion-selective diffusion channels induced underphosphate limitation
PhoE
Diffusion channel for small moleculesReceptor for phages Tula, T2
OmpF (1.2 nm)
Diffusion channel for small moleculesReceptor for phages Tulb, T4
OmpC (1.1 nm)
Diffusion channel for various metabolites includingmaltose
OmpB
Physiological RoleProtein Porin
Outer membrane proteins (OMPs) of Gram Negative Bacteria
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- A separate compartment between the cell membrane and outer membrane in Gram (-) bacteria
- Seen in electron micrographs as space but should be considered an aqueous compartment
- Activites: redox reactions osmotic regulation solute transport protein secretion hydrolysis
PERIPLASM
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PERIPLASM:
COMPONENTS AND FUNCTIONS1. Oligosaccharides – thought to be involved in the osmotic regulation
of the periplasm because their amounts decrease when thecells are grown in media of high osmolarity
2. Solute binding proteins – bind to solutes and deliver solutes to specific transporters in the membrane
3. Cytochrome – cyt c
4. Hydrolytic enzymes –degrade nutrients to smaller molecules thatcan be transported across the membrane by specifictransporter
5. Detoxifying agents – e.g. β-lactamase
6. TonB protein – required for the uptake of several solutes (iron siderophores, vit B12) that do not diffuse through the porin
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PERIPLASM: ACTIVITIES
• Protein transport
• Nutrient acquisition
• Protein folding
• Disulfide bond formation
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PERIPLASM IN GRAM POSITIVE CELLS?
Evidences:
• release of putative periplasmic proteins (distinctnucleases) in protoplasts of Bacillus subtilis • area outside the cell membrane of B. subtilis isbipartite (cryo-TEM)
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Archaeal Cell Walls
- Lacks peptidoglycan
- May contain polysaccharide, protein (S layer) orpseudopeptidoglycan
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Archaeal Cell Walls: Polysaccharide
• cell wall composed of glucose, glucuronic acid, acetate and galactosamine
• found in Methanosarcina spp.
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Archaeal Cell Walls: S-layer
- protein subunits arranged in a regular array on the cell surface
- found in extreme halophiles, methanogens and hyperthermophiles
Albers et al. Nature Reviews Microbiology advance online publication;published online 06 June 2006 | doi:10.1038/nrmicro1440
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Archaeal Cell Walls: S-layer
Archaeal Gram negativecell wall
Archaeal Gram positive cell wall
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Archaeal Cell Walls:Pseudopeptidoglycan
- also called pseudomurein
- consists of glycan units linked by peptides
- glycan units: N-acetylglucosamine N-acetyltalosaminuronic acid
- peptide bridge contains L-amino acids rather than D amino acids
NAT: N-acetylalosaminuronic acid
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Mollicutes: A Special Case
Mycoplasma; Spiroplasma Many are parasitic and pathogenic. small cell wall-less bacteria but possess
distinct morphologies Have internal protein cytoskeleton
that determines and maintains cell shape
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Spiroplasma
Williamson, D. L. 1974. J. Bacteriol. 117:904-906.